Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.
Activation of tyrosine kinases transmits extracellular cues to signal transduction cascades that result in a diverse set of adaptive events, while misregulation of tyrosine kinase signaling is prominent in many diseases. Clearly, the spatial and temporal dynamics of tyrosine kinase activity is fundamental to determining its diverse downstream effects.
Understanding neuronal plasticity, development, and function will increasingly depend on the ability to probe signaling events at the single cell and sub-cellular levels in living tissues and organisms. In the last twenty years, dye-based and genetically encoded calcium indicators that allow neuronal activity and calcium influx to be monitored in living neurons have substantially advanced our understanding of neuronal function. During the next twenty years genetically encoded tools for dissecting cell signaling in live cells will likely have even greater impact.
Initiation of intracellular signaling typically begins when transmembrane receptor molecules bind soluble or membrane attached ligands. Receptor tyrosine kinases (RTKs) are one of the most studied and prevalent classes of receptor proteins and include many molecules with prominent roles in neuronal development and plasticity including Trk and Eph receptors (Flanagan and Vanderhaeghen, 1998; Kaplan and Miller, 2000). Overall, there are approximately 20 families of these kinases that mediate events from cell differentiation, synapse formation, synaptic plasticity and cell survival. Precise activation of receptor TKs is important for specifying the biological outcome of their signaling while over-activation can result in diseases such as cancer. Normally, kinase activity of these proteins is induced by simple ligand induced dimerization (Trks) or multimerization (Ephs). However, we have little understanding of the fine-scale subcellular dynamic regulation of these molecules.
It is clear a need exists in the art for tyrosine kinase activity detection reagents. Ideally such reagents would be sensitive enough to correlate activity with specific tyrosine kinase action in real time.